Medical diagnostic tests in clinical laboratories commonly require stringent quality control as mandated by government agencies and standards organizations. The National Committee for Clinical Laboratory Standards (NCCLS) suggests accreditation guidelines that include calibrating equipment against control samples and performing tests of patient samples in tandem with consistent references (NCCLS, Villanova, Pa.). Other organizations, such as the Joint Commission on Accreditation of Healthcare Organizations (JCAHO) and the American Society for Clinical Pathology (ASCP) also recommend or mandate standardized clinical procedures often requiring updated (non-expired) and well-inventoried supplies of clinical reference reagents and controls (JCAHO, Washington D.C.; ASCP, Chicago, Ill.). Control references must be tested in conjunction with each test of a patient sample according to the Clinical Laboratory Improvement Act of 1988, which applies to over 175,000 laboratory entities (CLIA '88 is described at 42 C.F.R., parts 493.1-493.1850). The College of American Pathologists (CAP) and the American College of Medical Genetics (ACMG) also mandate comparison with references during each patient test (CAP, Northfield, Ill.; ACMG, Bethesda, Md.).
Clinical assays often involve either deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). For example, nucleic acid diagnostics may be performed to find infectious DNA or RNA from an invading virus or bacteria. Reference nucleic acids are typically used as chromatographic, spectroscopic, and/or spectrophotometric controls, for example during gel electrophoresis monitored by a laser “electric eye.”
DNA provides a template for generating bodily proteins using sequences of four DNA bases (adenine, thymine, guanine, and cytosine). The mutation of any one base in a single-stranded DNA sequence may be enough to form a defective allele resulting in disease. Many disease moieties thus have an underlying genetic etiology.
A gene is a length of DNA on a chromosome associated with some particular process or characteristic of an individual. A gene is conventionally considered a fundamental building block of heredity that determines observable characteristics, i.e., the “phenotype” of the individual organism. The underlying “chemical” genetic constitution of the individual is instead called its “genotype.”
Genes are observed to be lined up on human chromosomes in a sequential order. The sequential order of genes is the same for both members of a chromosome pair. Therefore genes occur in pairs (homologous genes). The two genes in a pair may occur in different forms called “alleles” and the phenotypic expression of one allele or the other in a pair depends on the types of alleles present. Mutations are the changes in the DNA sequence that may convert one allele to another. An individual who carries two of the same alleles is homozygous for that gene while an individual who has two different alleles for a gene pair is heterozygous for that gene. The occurrence of mutations that are deleterious to the normal expression of an allele may result in malfunction of that allele. The co-occurrence of a “normal” allele with a “mutated” (or abnormal) allele at the same gene in a heterozygous individual may result in a new (or disease) phenotype. In such a case, the mutated allele is described as acting in a dominant fashion over the normal allele. If the mutated allele does not cause any change in phenotype of the heterozygous individual, but causes a change (or disease) only when the individual is homozygous for that mutated allele, the mutated allele is described as acting in a recessive fashion compared to the normal allele. Thus, dominance and recessiveness describe the relative effect of gene expression of an allele when two distinct alleles occur together.
Carriers of genetic diseases typically carry a heterozygous recessive allele that includes a mutation capable of causing the disease. However, the mutated recessive allele may not be expressed in the carrier because its deleterious effect on the phenotype is masked by the normal (non-mutated) allele. Thus, a carrier may possess mutations in his genotype that can be passed down to descendents to cause the disease yet the carrier presents a normal phenotype (expressed characteristics) and is thus disease free. On the other hand, a person who experiences a genetically mediated disease may be a heterozygous “carrier” who has a mutated dominant allele for the disease. Still further, a person who experiences a disease may be a homozygous “carrier” with identical homologous genes that each has a mutation at a particular locus that causes the disease.
Given the number of different kinds of genetic diseases, the different possibilities for homozygous and heterozygous causation, and the need for both disease and carrier testing, maintaining recommended or mandated clinical supplies of high quality nucleic acid references and controls (hereinafter referred to as “reference nucleic acids” or just “references”) presents daunting challenges to genetics reference facilities and molecular diagnostic laboratories. As shown in FIG. 1, a reference nucleic acid 100 to be amplified for use as a test control comprises single-stranded or double-stranded reference RNA or DNA of known quantity and known quality within currently accepted tolerances. The ideal reference nucleic acid 100 to be amplified should resemble the patient sample to be tested as closely as possible and moreover, should be usable in all configurations of a given type of test. However, a reference nucleic acid 100 for clinical use may not be easily available in an adequate quantity and quality. Further, once an adequate quantity and quality of the reference nucleic acid 100 is obtained, the reference also needs to be reasonably easy to manufacture and store. These various requirements are difficult to meet because a single patient test often includes many diverse steps, such as polymerase chain reactions, enzymatic manipulations, sequencing reactions, hybridizations, electrophoreses, etc., each placing a different demand on the reference. Limited sources for obtaining a reference nucleic acid 100 to be amplified exacerbate a quality problem by causing a short supply leading to an increased likelihood that references of lower quality will be allowed in order to bolster the supply.
Typically, a reference nucleic acid 100 to be amplified originates from a human source 102, but if not available in sufficient quantity or not amenable to storage, then conventional chemical synthesis 104 may augment or replace the human source 102. Depending on the identity of the reference nucleic acid 100 to be amplified and its origin, various methods may be needed to refine, develop and increase the supply, each method yielding a product that may or may not have consistent quality with products yielded by other methods. A first conventional method 106 may merely isolate and purify the reference nucleic acid 100 to be amplified from a human source 102 and/or from conventional chemical synthesis 104. A second conventional method 108 may replicate the reference nucleic acid 100 to be amplified by cloning in a vector (e.g., a plasmid) and allowing a species of bacteria to propagate the vector. A third conventional method 110 may undertake amplification of a human-derived reference nucleic acid 100 in an automated cycler. Other conventional methods not reviewed here are represented by an “Nth” conventional method 112 that yields a variable, heterogeneous product. Most of these known methods yield products that may have inconsistent quality and/or stability, and many of the example methods are cumbersome and expensive, as well as dependent on starting materials from a human source 102.